PERSPECTIVE
Why Are tRNAs Overproduced in the Absence
of Maf1, a Negative Regulator of RNAP III,
Not Fully Functional?
Magdalena Boguta*
Department of Genetics, Institute of Biochemistry and Biophysics, Warsaw, Poland
* [email protected]
OPEN ACCESS
Citation: Boguta M (2015) Why Are tRNAs
Overproduced in the Absence of Maf1, a Negative
Regulator of RNAP III, Not Fully Functional? PLoS
Genet 11(12): e1005743. doi:10.1371/journal.
pgen.1005743
Editor: Anita Hopper, Ohio State University, UNITED
STATES
Published: December 31, 2015
Copyright: © 2015 Magdalena Boguta. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Funding: Magdalena Boguta is supported by the
National Science Centre (UMO-012/04/A/NZ1/
00052). The funders had no role in the preparation of
the article.
Competing Interests: The author has declared that
no competing interests exist.
tRNA biosynthesis in the eukaryotic cell is a multistep pathway, involving transcription, 5' and
3' end maturation, intron removal, and numerous modifications of nucleotides. Most of the
genes coding for each of the elements essential for tRNA biosynthetic activities were primarily
identified by genetic selection in yeast [1]. In these studies, the parental strain contained a
tRNA gene that had been converted to a nonsense suppressor. Given the appropriate genetic
background, phenotypic loss of suppression was used to select mutants producing non-functional tRNA. Among other proteins controlling tRNA biosynthesis, this approach led to identification of Maf1, a global repressor of tRNA transcription that is activated in response to stress.
The maf1-1 mutant was originally selected in a genetic screen for decreased efficiency of tRNA
suppressor SUP11 (tRNA Tyr/UAA) in budding yeast, Saccharomyces cerevisiae [2]. The role
of Maf1 has been suggested by tRNA accumulation in maf1Δ cells, observed regardless of the
repressive growth conditions [3]. An analogous decrease of tRNA-suppressor (tRNA Ser/
UCA) activity was detected for the maf1Δ mutant in Schizosaccharomyces pombe [4]. Interestingly, the effect of Maf1 on the efficiency of tRNA-mediated suppression is contrary to that
expected. Although one would assume that increased cellular tRNA levels should improve the
efficiency of tRNA-mediated nonsense suppression, data show the opposite is true.
Despite nearly two decades since the original discovery, the mechanism by which tRNA
accumulation in the maf1Δ mutant leads to the antisuppressor phenotype is still not understood. The simplest hypothesis is that tRNAs transcribed in maf1Δ cells are incompletely processed, hypomodified, or fail to be appropriately delivered to ribosomes. It is worth noting that
both primary transcripts and end-processed, intron-containing tRNA precursors were abnormally abundant in the absence of Maf1, and the nuclear export machinery was overloaded [5].
It was, however, unknown which processes in the tRNA maturation pathway were saturated by
the increased amounts of primary transcripts in cells lacking Maf1.
The current study by Arimbasseri and colleagues [4] solves a long-term conundrum: why
tRNAs overproduced in the absence of Maf1 are not fully functional. Their elegant work makes
a convincing case for the saturation of the dimethyltransferase Trm1 playing a crucial role in
the mechanism by which Maf1 affects tRNA suppression. By using tRNA-HydroSeq technique
to examine tRNA modification levels in S. pombe on the global scale, the authors have shown
that Trm1 substrates are not fully modified even in wild type cells. Further decrease of
Trm1-mediated G26 dimethylation on certain tRNAs was detected in the maf1Δ mutant. Consequently, hypomodification of G26 due to limited Trm1 reduces the activity of tRNA-suppressor-Ser/UCA and accounts for antisuppression. This hypothesis was validated by genetic
complementation of the antisuppressor phenotype of the maf1Δ mutant in fission yeast by
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Fig 1. A novel link between RNAP III transcription and tRNA modification. Transcription of tRNA
precursors by RNA polymerase III (RNAP III) in the nucleus is controlled by the general repressor Maf1. Initial
5' and 3' end maturation including addition of CCA sequence, takes place in the nucleus. Following m22G26
modification by Trm1 methyltransferase tethered to the inner nuclear membrane, tRNAs are exported to the
cytoplasm, charged, and directed to translation machinery. A previously unknown link connecting RNAP III
activity and m22G26 efficiency, due to a limiting amount of Trm1, is designated by a dashed line. Splicing of
intron-containing tRNA precursors and other modifications that can be added in each step of tRNA
biosynthesis are not presented on the scheme.
doi:10.1371/journal.pgen.1005743.g001
overproduced Trm1 [4]. Moreover, treatment with rapamycin or overexpression of Maf1
reduced tRNA transcription with increase in the m22G26 content of tRNA-suppressor-Ser/
UCA and its specific activity for suppression. Additionally, RNA polymerase III (RNAP III)
mutations associated with hypomyelinating leukodystrophy decreased tRNA transcription,
increased m22G26 efficiency, and reversed antisuppression. Taken together, these results demonstrate that increases or decreases in global RNAP III activity lead to inverse changes in the
efficiency of m22G26 modification of specific tRNAs. Thus, a previously unknown link connecting RNAP III activity and m22G26 efficiency is due to a limiting amount of Trm1 (Fig 1).
This link has been conserved through evolution, since the authors showed that the increase of
m22G26 content in specific tRNAs in response to starvation was detected in the human embryonic kidney. Moreover, increased production of Trm1 in S. cerevisiae maf1Δ cells led to reversal
of the antisuppression phenotype, as was also observed in fission yeast. In both these cases,
however, reversal of antisuppression by overproduced Trm1 is incomplete, suggesting that
other factors involved in tRNA maturation might also be saturated in the context of increased
tRNA synthesis in maf1Δ [4].
Why might tRNA modification by Trm1 be limited? A simple explanation could be that the
level of Trm1 protein is regulated by the growth conditions that affect tRNA transcription.
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This was, however, excluded experimentally, suggesting that Trm1 activity may be controlled
by a posttranslational mechanism. Another interesting possibility is that Trm1 modification
might be limited by tRNA retention time in the nucleus. In S. cerevisiae, Trm1 is tethered to
the inner nuclear membrane via a specific amino acid sequence tract [6]. Although nuclear residence may limit the time during which a nascent pre-tRNA transcript might have access to
acquire the m22G26 modification, retrograde RNA transport should theoretically allow iterative access to Trm1 [7]. Therefore, the mechanism by which cells maintain Trm1 activity in a
functionally limiting amount is unclear.
G26 resides at the junction between the D-stem and the anticodon stem, and its N2dimethylation, which interferes with normal Watson-Crick base pairing, may contribute to
prevention of tRNA misfolding. It is also notable that treatment of cells with 5-flurouracil
(5FU), which is incorporated into RNA, sensitizes S. cerevisiae to loss of genes that encode
tRNA modification enzymes whose nucleoside targets localize at or near the stems junction
and include gene-encoding Trm1 [8]. These observations, together with evidence that m22G26
can stabilize correctly folded anticodon stems [9], suggest that m22G26 may enhance tRNAspecific activity by improving the fit in the ribosome.
Although such a speculative role of Trm1 is beyond the scope of the current study, there are
examples of tRNA modification enzymes that affect posttranscriptional regulatory mechanisms. Trm9, a methyltransferase that catalyzes modification of wobble bases in the tRNA anticodon, enhances the translation of the class of transcripts overrepresented with specific
arginine and glutamic acid codons, which encode key damage response proteins [10]. Next,
modification of tRNA-Lys/UUU by an elongator is essential for efficient translation of stress
mRNAs [11]. Finally, loss of tRNA anticodon wobble uridine modification slows translation at
cognate codons, leading to widespread protein aggregation [12]. Because of its hierarchical substrate preference, convincingly documented by Arimbasseri and colleagues, Trm1 may also
contribute to maintaining proteome integrity.
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